MaC2H2-IDD regulates fruit softening and involved in softening disorder induced by cold stress in banana.

Chilling stress causes banana fruit softening disorder and severely impairs fruit quality. Various factors, such as transcription factors, regulate fruit softening. Herein, we identified a novel regulator, MaC2H2-IDD, whose expression is closely associated with fruit ripening and softening disorder. MaC2H2-IDD is a transcriptional activator located in the nucleus. The transient and ectopic overexpression of MaC2H2-IDD promoted "Fenjiao" banana and tomato fruit ripening. However, transient silencing of MaC2H2-IDD repressed "Fenjiao" banana fruit ripening. MaC2H2-IDD modulates fruit softening by activating the promoter activity of starch (MaBAM3, MaBAM6, MaBAM8, MaAMY3, and MaISA2) and cell wall (MaEXP-A2, MaEXP-A8, MaSUR14-like, and MaGLU22-like) degradation genes. DLR, Y1H, EMSA, and ChIP-qPCR assays validated the expression regulation. MaC2H2-IDD interacts with MaEBF1, enhancing the regulation of MaC2H2-IDD to MaAMY3, MaEXP-A2, and MaGLU22-like. Overexpressing/silencing MaC2H2-IDD in banana and tomato fruit altered the transcript levels of the cell wall and starch (CWS) degradation genes. Several differentially expressed genes (DEGs) were authenticated between the overexpression and control fruit. The DEGs mainly enriched biosynthesis of secondary metabolism, amino sugar and nucleotide sugar metabolism, fructose and mannose metabolism, starch and sucrose metabolism, and plant hormones signal transduction. Overexpressing MaC2H2-IDD also upregulated protein levels of MaEBF1. MaEBF1 does not ubiquitinate or degrade MaC2H2-IDD. These data indicate that MaC2H2-IDD is a new regulator of CWS degradation in "Fenjiao" banana and cooperates with MaEBF1 to modulate fruit softening, which also involves the cold softening disorder.

Discovery of gene regulation mechanisms associated with uniconazole-induced cold tolerance in banana using integrated transcriptome and metabolome analysis.

BACKGROUND: The gibberellic acid (GA) inhibitor, uniconazole, is a plant growth regulator commonly used in banana cultivation to promote dwarfing but also enhances the cold resistance in plants. However, the mechanism of this induced cold resistance remains unclear. RESULTS: We confirmed that uniconazole induced cold tolerance in bananas and that the activities of Superoxide dismutase and Peroxidase were increased in the uniconazole-treated bananas under cold stress when compared with the control groups. The transcriptome and metabolome of bananas treated with or without uniconazole were analyzed at different time points under cold stress. Compared to the control group, differentially expressed genes (DEGs) between adjacent time points in each uniconazole-treated group were enriched in plant-pathogen interactions, MAPK signaling pathway, and plant hormone signal transduction, which were closely related to stimulus-functional responses. Furthermore, the differentially abundant metabolites (DAMs) between adjacent time points were enriched in flavone and flavonol biosynthesis and linoleic acid metabolism pathways in the uniconazole-treated group than those in the control group. Temporal analysis of DEGs and DAMs in uniconazole-treated and control groups during cold stress showed that the different expression patterns in the two groups were enriched in the linoleic acid metabolism pathway. In addition to strengthening the antioxidant system and complex hormonal changes caused by GA inhibition, an enhanced linoleic acid metabolism can protect cell membrane stability, which may also be an important part of the cold resistance mechanism of uniconazole treatment in banana plants. CONCLUSIONS: This study provides information for understanding the mechanisms underlying inducible cold resistance in banana, which will benefit the production of this economically important crop.

Low temperature-induced regulatory network rewiring via WRKY regulators during banana peel browning.

Banana (Musa spp.) fruits, as typical tropical fruits, are cold sensitive, and lower temperatures can disrupt cellular compartmentalization and lead to severe browning. How tropical fruits respond to low temperature compared to the cold response mechanisms of model plants remains unknown. Here, we systematically characterized the changes in chromatin accessibility, histone modifications, distal cis-regulatory elements, transcription factor binding, and gene expression levels in banana peels in response to low temperature. Dynamic patterns of cold-induced transcripts were generally accompanied by concordant chromatin accessibility and histone modification changes. These upregulated genes were enriched for WRKY binding sites in their promoters and/or active enhancers. Compared to banana peel at room temperature, large amounts of banana WRKYs were specifically induced by cold and mediated enhancer-promoter interactions regulating critical browning pathways, including phospholipid degradation, oxidation, and cold tolerance. This hypothesis was supported by DNA affinity purification sequencing, luciferase reporter assays, and transient expression assay. Together, our findings highlight widespread transcriptional reprogramming via WRKYs during banana peel browning at low temperature and provide an extensive resource for studying gene regulation in tropical plants in response to cold stress, as well as potential targets for improving cold tolerance and shelf life of tropical fruits.

The impact of slow stomatal kinetics on photosynthesis and water use efficiency under fluctuating light.

Dynamic light conditions require continuous adjustments of stomatal aperture. The kinetics of stomatal conductance (gs) is hypothesized to be key to plant productivity and water use efficiency (WUE). Using step-changes in light intensity, we studied the diversity of light-induced gs kinetics in relation to stomatal anatomy in five banana genotypes (Musa spp.) and modeled the impact of both diffusional and biochemical limitations on photosynthesis (A). The dominant A limiting factor was the diffusional limitation associated with gs kinetics. All genotypes exhibited a strong limitation of A by gs, indicating a priority for water saving. Moreover, significant genotypic differences in gs kinetics and gs limitations of A were observed. For two contrasting genotypes, the impact of differential gs kinetics was further investigated under realistic diurnally fluctuating light conditions and at the whole-plant level. Genotype-specific stomatal kinetics observed at the leaf level was corroborated at whole-plant level by transpiration dynamics, validating that genotype-specific responses are still maintained despite differences in gs control at different locations in the leaf and across leaves. However, under diurnally fluctuating light conditions the impact of gs speediness on A and intrinsic (iWUE) depended on time of day. During the afternoon there was a setback in kinetics: absolute gs and gs responses to light were damped, strongly limiting A and impacting diurnal iWUE. We conclude the impact of differential gs kinetics depended on target light intensity, magnitude of change, gs prior to the change in light intensity, and particularly time of day.

A digital sensor to measure real-time leaf movements and detect abiotic stress in plants.

Plant and plant organ movements are the result of a complex integration of endogenous growth and developmental responses, partially controlled by the circadian clock, and external environmental cues. Monitoring of plant motion is typically done by image-based phenotyping techniques with the aid of computer vision algorithms. Here we present a method to measure leaf movements using a digital inertial measurement unit (IMU) sensor. The lightweight sensor is easily attachable to a leaf or plant organ and records angular traits in real-time for two dimensions (pitch and roll) with high resolution (measured sensor oscillations of 0.36 ± 0.53° for pitch and 0.50 ± 0.65° for roll). We were able to record simple movements such as petiole bending, as well as complex lamina motions, in several crops, ranging from tomato to banana. We also assessed growth responses in terms of lettuce rosette expansion and maize seedling stem movements. The IMU sensors are capable of detecting small changes of nutations (i.e. bending movements) in leaves of different ages and in different plant species. In addition, the sensor system can also monitor stress-induced leaf movements. We observed that unfavorable environmental conditions evoke certain leaf movements, such as drastic epinastic responses, as well as subtle fading of the amplitude of nutations. In summary, the presented digital sensor system enables continuous detection of a variety of leaf motions with high precision, and is a low-cost tool in the field of plant phenotyping, with potential applications in early stress detection.

MaMAPK3-MaICE1-MaPOD P7 pathway, a positive regulator of cold tolerance in banana.

BACKGROUND: Banana is a tropical fruit with a high economic impact worldwide. Cold stress greatly affects the development and production of banana. RESULTS: In the present study, we investigated the functions of MaMAPK3 and MaICE1 involved in cold tolerance of banana. The effect of RNAi of MaMAPK3 on Dajiao (Musa spp. 'Dajiao'; ABB Group) cold tolerance was evaluated. The leaves of the MaMAPK3 RNAi transgenic plants showed wilting and severe necrotic symptoms, while the wide-type (WT) plants remained normal after cold exposure. RNAi of MaMAPK3 significantly changed the expressions of the cold-responsive genes, and the oxidoreductase activity was significantly changed in WT plants, while no changes in transgenic plants were observed. MaICE1 interacted with MaMAPK3, and the expression level of MaICE1 was significantly decreased in MaMAPK3 RNAi transgenic plants. Over-expression of MaICE1 in Cavendish banana (Musa spp. AAA group) indicated that the cold resistance of transgenic plants was superior to that of the WT plants. The POD P7 gene was significantly up-regulated in MaICE1-overexpressing transgenic plants compared with WT plants, and the POD P7 was proved to interact with MaICE1. CONCLUSIONS: Taken together, our work provided new and solid evidence that MaMAPK3-MaICE1-MaPOD P7 pathway positively improved the cold tolerance in monocotyledon banana, shedding light on molecular breeding for the cold-tolerant banana or other agricultural species.

Identification of transcription factors interacting with a 1274 bp promoter of MaPIP1;1 which confers high-level gene expression and drought stress Inducibility in transgenic Arabidopsis thaliana.

BACKGROUND: Drought stress can severely affect plant growth and crop yield. The cloning and identification of drought-inducible promoters would be of value for genetically-based strategies to improve resistance of crops to drought. RESULTS: Previous studies showed that the MaPIP1;1 gene encoding an aquaporin is involved in the plant drought stress response. In this study, the promoter pMaPIP1;1, which lies 1362 bp upstream of the MaPIP1;1 transcriptional initiation site, was isolated from the banana genome..And the transcription start site(A) is 47 bp before the ATG. To functionally validate the promoter, various lengths of pMaPIP1;1 were deleted and fused to GUS to generate pMaPIP1;1::GUS fusion constructs that were then transformed into Arabidopsis to generate four transformants termed M-P1, M-P2, M-P3 and M-P4.Mannitol treatment was used to simulate drought conditions. All four transformants reacted well to mannitol treatment. M-P2 (- 1274 bp to - 1) showed the highest transcriptional activity among all transgenic Arabidopsis tissues, indicating that M-P2 was the core region of pMaPIP1;1. This region of the promoter also confers high levels of gene expression in response to mannitol treatment. Using M-P2 as a yeast one-hybrid bait, 23 different transcription factors or genes that interacted with MaPIP1;1 were screened. In an dual luciferase assay for complementarity verification, the transcription factor MADS3 positively regulated MaPIP1;1 transcription when combined with the banana promoter. qRT-PCR showed that MADS3 expression was similar in banana leaves and roots under drought stress. In banana plants grown in 45% soil moisture to mimic drought stress, MaPIP1;1 expression was maximized, which further demonstrated that the MADS3 transcription factor can synergize with MaPIP1;1. CONCLUSIONS: Together our results revealed that MaPIP1;1 mediates molecular mechanisms associated with drought responses in banana, and will expand our understanding of how AQP gene expression is regulated. The findings lay a foundation for genetic improvement of banana drought resistance.

Genome-Wide Identification and Characterization of the NAC Transcription Factor Family in Musa Acuminata and Expression Analysis during Fruit Ripening.

Banana (Musa acuminata, AAA group) is a representative climacteric fruit with essential nutrients and pleasant flavors. Control of its ripening determines both the fruit quality and the shelf life. NAC (NAM, ATAF, CUC2) proteins, as one of the largest superfamilies of transcription factors, play crucial roles in various functions, especially developmental processes. Thus, it is important to conduct a comprehensive identification and characterization of the NAC transcription factor family at the genomic level in M. acuminata. In this article, a total of 181 banana NAC genes were identified. Phylogenetic analysis indicated that NAC genes in M. acuminata, Arabidopsis, and rice were clustered into 18 groups (S1-S18), and MCScanX analysis disclosed that the evolution of MaNAC genes was promoted by segmental duplication events. Expression patterns of NAC genes during banana fruit ripening induced by ethylene were investigated using RNA-Seq data, and 10 MaNAC genes were identified as related to fruit ripening. A subcellular localization assay of selected MaNACs revealed that they were all localized to the nucleus. These results lay a good foundation for the investigation of NAC genes in banana toward the biological functions and evolution.

MaMPK2 enhances MabZIP93-mediated transcriptional activation of cell wall modifying genes during banana fruit ripening.

Transcriptional regulation is an essential molecular machinery in controlling gene expression in diverse plant developmental processes including fruit ripening. This involves the interaction of transcription factors (TFs) and promoters of target genes. In banana, although a number of fruit ripening-associated TFs have been characterized, their number is relatively small. Here we identified a nuclear-localized basic leucine zipper (bZIP) TF, MabZIP93, associated with banana ripening. MabZIP93 activated cell wall modifying genes MaPL2, MaPE1, MaXTH23 and MaXGT1 by directly binding to their promoters. Transient over-expression of MabZIP93 in banana fruit resulted in the increased expression of MaPL2, MaPE1, MaXTH23 and MaXGT1. Moreover, a mitogen-activated protein kinase MaMPK2 and MabZIP93 were found to interact with MabZIP93. The interaction of MabZIP93 with MaMPK2 enhanced MabZIP93 activation of cell wall modifying genes, which was likely due to the phosphorylation of MabZIP93 mediated by MaMPK2. Overall, this study shows that MaMPK2 interacts with and phosphorylates MabZIP93 to promote MabZIP93-mediated transcriptional activation of cell wall modifying genes, thereby expanding our understanding of gene networks associated with banana fruit ripening.

Genome-wide characterization of a SRO gene family involved in response to biotic and abiotic stresses in banana (Musa spp.).

BACKGROUND: Banana (Musa spp.) is one of the world's most important fruits and its production is largely limited by diverse stress conditions. SROs (SIMILAR TO RCD-ONE) have important functions in abiotic stress resistance and development of plants. They contain a catalytic core of the poly(ADP-ribose) polymerase (PARP) domain and a C-terminal RST (RCD-SRO-TAF4) domain. In addition, partial SROs also include an N-terminal WWE domain. Although a few of SROs have been characterized in some model plants, little is known about their functions in banana, especially in response to biotic stress. RESULTS: Six MaSRO genes in banana genome were identified using the PARP and RST models as a query. Phylogenetic analysis showed that 77 SROs from 15 species were divided into two structurally distinct groups. The SROs in the group I possessed three central regions of the WWE, PARP and RST domains. The WWE domain was lacking in the group II SROs. In the selected monocots, only MaSROs of banana were present in the group II. Most of MaSROs expressed in more than one banana tissue. The stress- and hormone-related cis-regulatory elements (CREs) in the promoter regions of MaSROs supported differential transcripts of MaSROs in banana roots treated with abiotic and biotic stresses. Moreover, expression profiles of MaSROs in the group I were clearly distinct with those observed in the group II after hormone treatment. Notably, the expression of MaSRO4 was significantly upregulated by the multiple stresses and hormones. The MaSRO4 protein could directly interact with MaNAC6 and MaMYB4, and the PARP domain was required for the protein-protein interaction. CONCLUSIONS: Six MaSROs in banana genome were divided into two main groups based on the characteristics of conserved domains. Comprehensive expression analysis indicated that MaSROs had positive responses to biotic and abiotic stresses via a complex interaction network with hormones. MaSRO4 could interact directly with MaNAC6 and MaMYB4 through the PARP domain to regulate downstream signaling pathway.

Bananas tackling drought and heat - with DREBs and more.

With the changing climate, crops are facing mounting threats from multiple abiotic stresses, and studies that assess the response of plants to combinations, rather than to individual, abiotic stresses are becoming increasingly relevant. Bananas are one of the most globally important and popular food crops and their production is threatened by increasing heat and diminishing rainfall in tropical and subtropical regions. In pursuit of effective stress management strategies, Jangale et al. (2019) look into the physiological and molecular responses of banana plants to combined heat and drought stresses.

Independent and combined abiotic stresses affect the physiology and expression patterns of DREB genes differently in stress-susceptible and resistant genotypes of banana.

In tropics, combined stresses of drought and heat often reduce crop productivity in plants like Musa acuminata L. We compared responses of two contrasting banana genotypes, namely the drought-sensitive Grand Nain (GN; AAA genome) and drought tolerant Hill banana (HB; AAB genome) to individual drought, heat and their combination under controlled and field conditions. Drought and combined drought and heat treatments caused greater reduction in leaf relative water content and greater increase in ion leakage and H2 O2 content in GN plants, especially in early stages, while the responses were more pronounced in HB at later stages. A combination of drought and heat increased the severity of responses. Real-time expression patterns of the A-1 and A-2 group DEHYDRATION-RESPONSIVE ELEMENT BINDING (DREB) genes revealed greater changes in expression in leaves of HB plants for both the individual stresses under controlled conditions compared to GN plants. A combination of heat and drought, however, activated most DREB genes in GN but surprisingly suppressed their expression in HB in controlled and field conditions. Its response seems correlated to a better stomatal control over transpiration in HB and a DREB-independent pathway for the more severe combined stresses unlike in GN. Most of the DREB genes had abscisic acid (ABA)-responsive elements in their promoters and were also activated by ABA suggesting at least partial dependence on ABA. This study provides valuable information on physiological and molecular responses of the two genotypes to individual and combined drought and heat stresses.

Differential proteomic analysis reveals the mechanism of Musa paradisiaca responding to salt stress.

Salinity is one of the most important abiotic stresses, which affects the yield and quality of banana (Musa paradisiaca). To understand the salinity tolerance mechanisms of banana, the iTRAQ technique is employed to reveal the proteomic response of Brazil banana under different durations of 60 mmol/L NaCl stress. We have identified 77 DEPs and classified them into nine functional categories, compared with control (0 mmol/L NaCl treatment). The four major categories involve protein synthesis and degradation, photosynthesis, defense response, and energy and carbohydrate metabolism. The results indicate that photosynthesis, protein synthesis and degradation, lipid metabolism and secondary metabolism are promoted to limit damage to a repairable level. The accumulation of ROS under salt stress is harmful to cells and causes up-regulation of antioxidant systems. Furthermore, to cope with cells injured by salt stress, PCD is used to remove the damaged. Additionally, the cytoskeleton can play an important role in maintaining cellular and redox homeostasis. Different categories of functional proteins by changing the abundance ratio shows that plants have different mechanisms of response to salinity. Conclusively, Function of the observed changes in protein expression objective is to establish a new metabolic process of steady-state balance. To my knowledge, this is the first report that investigates responses of M. paradisiaca to salt stress by proteomic analysis.

Cold-inducible MaC2H2s are associated with cold stress response of banana fruit via regulating MaICE1.

KEY MESSAGE: MaC2H2s are involved in cold stress response of banana fruit via repressing the transcription of MaICE1. Although C2H2 zinc finger proteins have been found to be involved in banana fruit ripening through transcriptional controlling of ethylene biosynthetic genes, their involvement in cold stress of banana remains elusive. In this study, another C2H2-ZFP gene from banana fruit was identified, which was named as MaC2H2-3. Gene expression analysis revealed that MaC2H2-1, MaC2H2-2 and MaC2H2-3 were cold inducible in the peel of banana during low temperature storage. MaC2H2-3 functions as a transcriptional repressor and localizes predominantly in nucleus. Particularly, promoters of MaC2H2-2 and MaC2H2-3 were noticeably activated by cold as well, further indicating the potential roles of C2H2 in cold stress of banana. Moreover, MaC2H2-2 and MaC2H2-3 significantly repressed the transcription of MaICE1, a key component in cold signaling pathway. Overall, these findings suggest that MaC2H2s may take part in controlling cold stress of banana through suppressing the transcription of MaICE1, providing new insight of the regulatory basis of C2H2 in cold stress.

A banana NAC transcription factor (MusaSNAC1) impart drought tolerance by modulating stomatal closure and H2O2 content.

KEY MESSAGE: MusaSNAC1 function in H2O2 mediated stomatal closure and promote drought tolerance by directly binding to CGT[A/G] motif in regulatory region of multiple stress-related genes. Drought is a abiotic stress-condition, causing reduced plant growth and diminished crop yield. Guard cells of the stomata control photosynthesis and transpiration by regulating CO2 exchange and water loss, thus affecting growth and crop yield. Roles of NAC (NAM, ATAF1/2 and CUC2) protein in regulation of stress-conditions has been well documented however, their control over stomatal aperture is largely unknown. In this study we report a banana NAC protein, MusaSNAC1 which induced stomatal closure by elevating H2O2 content in guard cells during drought stress. Overexpression of MusaSNAC1 in banana resulted in higher number of stomata closure causing reduced water loss and thus elevated drought-tolerance. During drought, expression of GUS (ß-glucuronidase) under P MusaSNAC1 was remarkably elevated in guard cells of stomata which correlated with its function as a transcription factor regulating stomatal aperture closing. MusaSNAC1 is a transcriptional activator belonging to SNAC subgroup and its 5'-upstream region contain multiple Dof1 elements as well as stress-associated cis-elements. Moreover, MusaSNAC1 also regulate multiple stress-related genes by binding to core site of NAC-proteins CGT[A/G] in their 5'-upstream region. Results indicated an interesting mechanism of drought tolerance through stomatal closure by H2O2 generation in guard cells, regulated by a NAC-protein in banana.

Sulfoxidation Regulation of Musa acuminata Calmodulin (MaCaM) Influences the Functions of MaCaM-Binding Proteins.

Sulfoxidation of methionine in proteins by reactive oxygen species can cause conformational alteration or functional impairment, and can be reversed by methionine sulfoxide reductase (Msr). Currently, only a few potential Msr substrates have been confirmed in higher plants. Here, we investigated Msr-mediated sulfoxidation regulation of calmodulin (CaM) and its underlying biological significance in relation to banana fruit ripening and senescence. Expression of MaCaM1 and MaMsrA7 was up-regulated with increased ripening and senescence. We verified that MaCaM1 interacts with MaMsrA7 in vitro and in vivo, and sulfoxidated MaCaM1 could be partly repaired by MaMsrA7 (MaMsrA7 reduces oxidized residues Met77 and Met110 in MaCaM1). Furthermore, we investigated two known CaM-binding proteins, catalase (MaCAT1) and MaHY5-1. MaHY5-1 acts as a transcriptional repressor of carotenoid biosynthesis-related genes (MaPSY1, MaPSY2 and MaPSY3) in banana fruit. MaCaM1 could enhance the catalytic activity of MaCAT1 and the transcriptional repression activity of MaHY5-1 toward MaPSY2. Mimicked sulfoxidation in MaCaM1 did not affect the physical interactions of the protein with MaHY5-1 and MaCAT1, but reduced the catalytic activity of MaCAT1 and the transcriptional repression activity of MaHY5-1. Our data suggest that sulfoxidation modification in MaCaM1 by MaMsrA7 regulates antioxidant response and gene transcription, thereby being involved in regulation of ripening and senescence of banana fruit.

High-throughput sequencing of small RNAs revealed the diversified cold-responsive pathways during cold stress in the wild banana (Musa itinerans).

BACKGROUND: Cold stress is one of the most severe abiotic stresses affecting the banana production. Although some miRNAs have been identified, little is known about the role of miRNAs in response to cold stress in banana, and up to date, there is no report about the role of miRNAs in the response to cold stress in the plants of the cultivated or wild bananas. RESULT: Here, a cold-resistant line wild banana (Musa itinerans) from China was used to profile the cold-responsive miRNAs by RNA-seq during cold stress. Totally, 265 known mature miRNAs and 41 novel miRNAs were obtained. Cluster analysis of differentially expressed (DE) miRNAs indicated that some miRNAs were specific for chilling or 0 °C treated responses, and most of them were reported to be cold-responsive; however, some were seldom reported to be cold-responsive in response to cold stress, e.g., miR395, miR408, miR172, suggesting that they maybe play key roles in response to cold stress. The GO and KEGG pathway enrichment analysis of DE miRNAs targets indicated that there existed diversified cold-responsive pathways, and miR172 was found likely to play a central coordinating role in response to cold stress, especially in the regulation of CK2 and the circadian rhythm. Finally, qPCR assays indicated the related targets were negatively regulated by the tested DE miRNAs during cold stress in the wild banana. CONCLUSIONS: In this study, the profiling of miRNAs by RNA-seq in response to cold stress in the plants of the wild banana (Musa itinerans) was reported for the first time. The results showed that there existed diversified cold-responsive pathways, which provided insight into the roles of miRNAs during cold stress, and would be helpful for alleviating cold stress and cold-resistant breeding in bananas.

Overexpression of a Novel ROP Gene from the Banana (MaROP5g) Confers Increased Salt Stress Tolerance.

Rho-like GTPases from plants (ROPs) are plant-specific molecular switches that are crucial for plant survival when subjected to abiotic stress. We identified and characterized 17 novel ROP proteins from Musa acuminata (MaROPs) using genomic techniques. The identified MaROPs fell into three of the four previously described ROP groups (Groups II⁻IV), with MaROPs in each group having similar genetic structures and conserved motifs. Our transcriptomic analysis showed that the two banana genotypes tested, Fen Jiao and BaXi Jiao, had similar responses to abiotic stress: Six genes (MaROP-3b, -5a, -5c, -5f, -5g, and -6) were highly expressed in response to cold, salt, and drought stress conditions in both genotypes. Of these, MaROP5g was most highly expressed in response to salt stress. Co-localization experiments showed that the MaROP5g protein was localized at the plasma membrane. When subjected to salt stress, transgenic Arabidopsis thaliana overexpressing MaROP5g had longer primary roots and increased survival rates compared to wild-type A. thaliana. The increased salt tolerance conferred by MaROP5g might be related to reduced membrane injury and the increased cytosolic K⁺/Na⁺ ratio and Ca2+ concentration in the transgenic plants as compared to wild-type. The increased expression of salt overly sensitive (SOS)-pathway genes and calcium-signaling pathway genes in MaROP5g-overexpressing A. thaliana reflected the enhanced tolerance to salt stress by the transgenic lines in comparison to wild-type. Collectively, our results suggested that abiotic stress tolerance in banana plants might be regulated by multiple MaROPs, and that MaROP5g might enhance salt tolerance by increasing root length, improving membrane injury and ion distribution.

Physiological, molecular and ultrastructural analyses during ripening and over-ripening of banana (Musa spp., AAA group, Cavendish sub-group) fruit suggest characteristics of programmed cell death.

BACKGROUND: Programmed cell death (PCD) is a part of plant development that has been studied for petal senescence and vegetative tissue but has not been thoroughly investigated for fleshy fruits. The purpose of this research was to examine ripening and over-ripening in banana fruit to determine if there were processes in common to previously described PCD. RESULTS: Loss of cellular integrity (over 40%) and development of senescence related dark spot (SRDS) occurred after day 8 in banana peel. Nuclease and protease activity in the peel increased during ripening starting from day 2, and decreased during over-ripening. The highest activity was for proteases and nucleases with apparent molecular weights of 86 kDa and 27 kDa, respectively. Images of SRDS showed shrinkage of the upper layers of cells, visually suggesting cell death. Decrease of electron dense areas was evident in TEM micrographs of nuclei. CONCLUSION: This study shows for the first time that ripening and over-ripening of banana peel share physiological and molecular processes previously described in plant PCD. SRDS could represent a morphotype of PCD that characterizes a structural and biochemical failure in the upper layers of the peel, thereafter spreading to lower and adjacent layers of cells. © 2017 Society of Chemical Industry.

The transcriptional regulatory network mediated by banana (Musa acuminata) dehydration-responsive element binding (MaDREB) transcription factors in fruit ripening.

Fruit ripening is a complex, genetically programmed process involving the action of critical transcription factors (TFs). Despite the established significance of dehydration-responsive element binding (DREB) TFs in plant abiotic stress responses, the involvement of DREBs in fruit ripening is yet to be determined. Here, we identified four genes encoding ripening-regulated DREB TFs in banana (Musa acuminata), MaDREB1, MaDREB2, MaDREB3, and MaDREB4, and demonstrated that they play regulatory roles in fruit ripening. We showed that MaDREB1-MaDREB4 are nucleus-localized, induced by ethylene and encompass transcriptional activation activities. We performed a genome-wide chromatin immunoprecipitation and high-throughput sequencing (ChIP-Seq) experiment for MaDREB2 and identified 697 genomic regions as potential targets of MaDREB2. MaDREB2 binds to hundreds of loci with diverse functions and its binding sites are distributed in the promoter regions proximal to the transcriptional start site (TSS). Most of the MaDREB2-binding targets contain the conserved (A/G)CC(G/C)AC motif and MaDREB2 appears to directly regulate the expression of a number of genes involved in fruit ripening. In combination with transcriptome profiling (RNA sequencing) data, our results indicate that MaDREB2 may serve as both transcriptional activator and repressor during banana fruit ripening. In conclusion, our study suggests a hierarchical regulatory model of fruit ripening in banana and that the MaDREB TFs may act as transcriptional regulators in the regulatory network.